In the flower industry, flowers having a new trait are always highly valued. In particular, the development of plants having a different “color”, the most important trait of flowers, is industrially very important, and so far flowers of a variety of colors have been developed by cultivar improvement using classical breeding methods. With these methods are effective in cultivar improvement, there are restrictions on the gene pool inherent to each plant, and thus the methods can be applied to gene resources owned by closely related species that are amenable to breeding. For example, despite long years of breeding efforts, no purple to blue varieties for roses, carnations, chrysanthemums or lilies, no bright red varieties for gentians or irises, and no yellow varieties for geranium or morning glories have been created.
Flower color results from four types of pigments, i.e., flavonoids, carotenoids, chlorophylls, and betalains. Among them, flavonoids contribute to a variety of colors such as yellow, red, purple and blue. The group of pigments that develop red, purple and blue colors is collectively termed anthocyanins, and the diversity of anthocyanin structures is one reason for the wide variety of flower colors. Considering the biosynthetic pathway, anthocyanins are roughly divided into three groups depending on the aglycon structure. Bright red-colored flowers such as carnation and geranium often contain pelargonidin-type anthocyanins, and blue- and purple-colored flowers often contain delphinidin-type anthocyanins. The absence of blue or purple varieties of roses, carnations, chrysanthemums and lilies is because they have no ability of synthesizing the delphinidin-type anthocyanins.
In order for flowers to have a blue color, in addition to the accumulation of delphinidins, it has been thought that either one of the following is required: (i) the modification of anthocyanins with one or a plurality of aromatic acyl group(s), (ii) coexistence of anthocyanins with copigments such as flavones and flavonols, (iii) coexistence of anthocyanins with iron ions or aluminum ions, (iv) the increase in pH of anthocyanin-localized vacuoles from neutral to weak alkali, and (v) complex formation by anthocyanins, copigments and metal ions (such anthocyanins are termed metalloanthocyanins) (Non-patent document 1 below).
Biosynthesis of flavonoids and anthocyanins has been well studied, and relevant biosynthetic enzymes and genes encoding them have been identified (see Non-patent document 2, FIG. 1 below). For example, genes of flavonoid 3′,5′-hydroxylase (F3′S′H), which hydroxylates the B ring of flavonoids required in delphinidin biosynthesis, have been obtained from many plants. Also, by introducing these F3′S′H genes into carnations (see Patent document 1 below), roses (see Non-patent document 3 and Patent documents 2 and 3 below), and chrysanthemums (see Patent document 4 below), gene recombinant plants in which delphinidins are accumulated in petals and flower color is changed to blue have been generated (see Non-patent document 4 below). Such carnations and roses are commercially available.
Flavones, a family of organic compounds, are cyclic ketones of flavane derivatives. In a narrower sense, it indicates 2,3-didehydroflavan-4-one, a compound having a chemical formula C15H10O2 and a molecular weight of 222.24. In a broader sense, derivatives belonging to flavanes are termed “flavone”. Flavonesas defined in the broader sense (flavones) constitute one category of flavonoids. Those flavonoids that have the flavone structure as the basic skeleton and have no hydroxyl groups at the 3-position are classified into “flavones”. Representative examples of “flavones” include apigenin (4′,5,7-trihydroxyflavone) and luteolin (3′,4′,5,7-tetrahydroxyflavone). As used herein the term “flavone” refers to a flavone as defined in the broader sense, i.e., a derivative belonging to flavone.
Genes of flavone synthase (FNS) required for flavone biosynthesis have also been obtained from many plants. Flavone, when coexistent with anthocyanin, is known to have an effect of making the color of anthocyanin bluer, and these FNS genes attracted attention in the modification of flower colors. By introducing the FNS gene together with F3′5′H into roses having no ability of synthesizing flavones, the flower petals accumulated delphinidin simultaneously with the accumulation of flavone, making flower color bluer (see Patent document 5 below). Since flavone absorbsan ultraviolet ray in addition to making flower color bluer, it protects plants against the ultraviolet ray or serves as a signal to vision of insects in insect-pollinated flowers. Flavone is also involved in interaction with soil microorganisms. Furthermore, flavone is used in materials for foods or cosmetics as ingredients good for health. For example, flavone is said to have an anti-cancer effect, and it has also been demonstrated that by taking flavone-rich food materials, cancer can be treated or prevented.
Genes that modify anthocyanin and flavone have also been obtained from many plants. There are glycosyltransferase, acyl transferase, methyl transferase etc., and, among them, glycosyltransferase (GT) that catalyzes glycosylation is described herein. For example, genes encoding a protein having an activity of transferring glucose to the hydroxyl group at the 3-position of anthocyanin have been isolated from gentian, perilla, petunia, rose, antirrhinum and the like (see Non-patent documents 4 to 6 and Patent Document 6). Genes encoding a protein having an activity of transferring glucose to a hydroxyl group at the 5-position of anthocyanin have been isolated from perilla, petunia, rose, gentian, verbena, torenia and the like (see Non-patent documents 5 to 7, and Patent document 7 below). A gene encoding a protein having an activity of transferring glucose to the hydroxyl group at the 7-position of flavone has been isolated from arabidopsis (see Non-patent document 8 below). A gene encoding a protein having an activity of transferring glucose to the hydroxyl group at the 7-position of baicalin has been isolated from Scutellaria baicalensis, and it is also reported that a protein obtained by expressing the gene in Escherichia coli catalyzes a reaction that exhibits an activity of transferring glucose to the hydroxyl group at the 7-position of flavonoid (see Non-patent document 9 below). A gene encoding a protein having an activity of transferring glucose to the hydroxyl group at the 3′-position of anthocyanin has been isolated from gentian, butterfly pea, and cineraria (see Patent document 8 below). Also, a gene encoding a protein having an activity of transferring glucose to hydroxyl groups at two different positions on the A and C rings of anthocyanin has been isolated from rose (see Patent document 9 below). A gene encoding a protein having an activity of transferring glucose to hydroxyl groups at two different positions of the B ring of anthocyanin has been isolated from butterfly pea (see Patent document 10 below).
While the glycosyltransferases mentioned above rely on UDP-glucose as a glycosyl donor, a glycosyltransferase whose glycosyl donor is acyl glucose has been identified recently. A gene encoding a protein having an activity of transferring glucose to the hydroxyl group at the 5-position of anthocyanin-3 glucoside has been isolated from carnation, and a gene encoding a protein having an activity of transferring glucose to a hydroxyl group at the 7-position has been isolated from delphinium (see, Non-patent document 10 below).
Thus, a multitude of proteins having an activity of transferring glucose to various hydroxyl groups are known as glycosyltransferases.
However, it is believed that there are still many glycosyltransferases of which functions have not been identified. For example, a gene encoding a protein having an activity of transferring a glycosyl to the 4′-position of a flavonoid, or a gene encoding a protein having an activity of transferring glycosyl sequentially to hydroxyl groups at two sites on the A and B rings of a flavonoid has not been identified yet. It is reported that a protein obtained by expressing a glycosyltransferase gene derived from Livingstone daisy in Escherichia coli exhibits an activity of transferring glucose to either one of the hydroxyl groups at the 4′-position and the 7-position of a flavonoid, but the original activity of the glycosyltransferase in plants is to transfer glucose to the hydroxyl group at the 5-position of betanidine (see Non-patent document 11 below).
A metalloanthocyanins, which is represented by pigments of Commelina, Centaurea, Salvia, and Nemophila, is composed of six molecules of anthocyanin, six molecules of flavone, and two metal atoms, which components aggregate to form a stable blue pigment (see FIG. 2, Non-patent document 1). For example, anthocyanin of Nemophila is composed of nemophilin (see FIG. 3), malonyl apigenin 4′,7-diglucoside (see FIG. 4), Mg2+ and Fe3+. Metalloanthocyanin of Salvia is composed of cyanosalvianin (see FIG. 5), and apigenin 4′,7-diglucoside (see FIG. 6) and Mg2+. Studies so far have demonstrated that in all blue flowers forming metalloanthocyanins, flavone in which a glycosyl has been added to both of the hydroxyl groups at the 4′-position and the 7-position, and the glycosyl added to the flavone has been shown to play an important role in molecular recognition in metalloanthocyanin formation. The glycosyl coordinated at the 4′-position of a flavone is important in molecular recognition during the formation, and the glycosyl at the 7-position has been indicated to contribute to its stability (see Non-patent document 1 below). Only after the addition of these two glycosyls, metalloanthocyanin is formed thereby expressing a beautiful blue color. In Dutch iris petals, flavone in which a glycosyl has been added to the 4′-position is contained. Since the addition of two glycosyls to a flavone leads to increased solubility and altered properties, the expansion of uses as materials for health food products, pharmaceutical products and cosmetic products can be expected.